Adoptive cell transfer (ACT) has potential clinical applications and is an active area of tumor immunotherapy research. Cytotoxic T lymphocytes (CTLs) are effector cells of the immune system and mediate tumor destruction.1 Tumor vaccines increase the number of tumorspecific CTLs, but eventually, the number of CTLs reaches a stationary phase, due mainly to the presence of specific and nonspecific immune regulatory networks. This limits the amplification of CTLs in vivo, whereas in vitro, CTLs evade the regulatory networks and amplification can be realized.2 The factors influencing adoptive immunotherapy include the number of cells infused, tumor specificity, tumor affinity, the cellular phenotype of tumors, the survival time of the effector cells, and the in vivo microenvironment. How to induce highly efficient CTLs has become a new topic in cancer immunotherapy.
In the present study, effector T cells were derived from C57BL/6 peripheral blood, naive T cells (TN), and pooled T lymphocytes (TP) sorted by negative selection. Tumoricidal efficiencies of the CTLs derived from naive and pooled T lymphocytes were evaluated to explore an expansion scheme for generating more efficient effector cells for ACT immunotherapy.
Cell lines and mice
The mouse B16 melanoma cell line was purchased from the Shanghai Institute of Cell Biology (Shanghai, China). The cells were cultivated in RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal calf serum (FCS) (HyClone Inc., Logan, UT, USA), 100 g/ml streptomycin, and 100 IU/ml penicillin with 5% CO2 at 37°C. Six-week-old C57BL/6 female mice were purchased from Beijing HFK Bioscience Co., Ltd (Beijing, China). All mice were bred and housed at the Fourth Hospital of Hebei Medical University Laboratory Animal Research Center (Shijiazhuang, Hebei Province, China). Experiments were carried out according to the guidelines of the Animal Ethics Committee of Hebei Medical University.
Tumor models and CTX peritoneal injection
C57BL/6 mice bearing B16 melanoma cells were used as tumor models. All C57BL/6 mice were injected subcutaneously in the back with 1×105 B16 melanoma cells. On the day of visible tumor appearance, a dose of 100 mg/kg cyclophosphamide (CTX) was injected peritoneally to interfere with tumor immunosuppression.
At various time points after CTX injection, spleens were removed and single-cell suspensions were prepared. Cells were stained for CD4+CD25+Foxp3+ using the Mouse Regulatory T cell Staining Kit (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions. All analyses were performed using BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), and the relevant data were analyzed using FlowJo software (version 6.4.7; Tree Star, Ashland, OR, USA). The proportion of splenic CD4+CD25+Foxp3+/CD4+ T cells observed was standard for Tregs.
Detection of tumor growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) by enzyme-linked immunosorbent assay (ELISA)
TGF-β1 and IL-10 serum concentrations were tested using specific ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol.
Dendritic cell generation
Bone marrow-derived dendritic cells (DCs) were separated from C57BL/6 femurs.3 Briefly, erythrocyte-depleted bone marrow cells (1×106 cells/ml) were cultured in complete medium (RPMI 1640 supplemented with 10% FCS, 100 mg/ml streptomycin, and 1 IU/ml penicillin) supplemented with 20 ng/ml rmIL-4 and 20 ng/ml rmGM-CSF (PeproTech, Rocky Hill, NJ, USA) with 5% CO2 at 37°C. Half of the media was removed on the third and fifth day, respectively. rmTNF-α (15 ng/ml; PeproTech) was added to stimulate immature DCs to differentiate into mature DCs on Day 6 for 24 hours. Mature DCs were analyzed by Fluorescence Activated Cell Sorter (FACS) by staining with fluorescein isothiocyanate (FITC) anti-mouse CD86, phycoerythrin (PE) anti-mouse major histocompatibility complex-II (MHC-II), and PE anti-mouse CD11 (eBioscience). The lysate produced by repeated freezethaw of B16 cells was then added to the DC culture on Day 7 at a ratio of five DCs to one tumor cell and continuously incubated for 48 hours. The tumor lysates pulsed with DCs (TL-DCs) were then harvested.
Negative selection of T cells and generation of tumorspecific CTLsin vitro
Erythrocyte-depleted splenic suspensions were harvested from C57BL/6 mice. Pooled T cells, naive CD8+T cells, and naive CD4+T cells were negatively selected from the same splenocyte suspension using the MagCellect Mouse CD3+T Cell, Naive CD8+T Cell, and Mouse Naive CD4+T Cell Isolation Kits (R&D Systems), respectively, according to the manufacturer's instructions. Cells were cultured in complete medium supplemented with 20 ng/ml recombined murine interleukin-2 (rmIL-2) (PeproTech) for 2 days. T cells were counted using a FACScan analyzer and stained with PE-Cy5 anti-mouse CD3, FITC anti-mouse CD8, FITC anti-mouse CD4, PE anti-mouse CD62L (L-selectin), and PE-Cy5 anti-human/mouse CD44 (eBioscience). Pooled T cells and the mixture of selected naive CD8+ and CD4+ T cells (CD4+ T cell:CD8+ T ratio=2:1) were cultured in complete medium. The TL-DCs were added to the culture systems on Day 3 (T cells: DC, 10:1) supplemented with rmIL-2 (20 ng/ml) and continuously incubated at 37°C for 72 hours. Dendritic cell-induced cytotoxic T lymphocytes were harvested for adoptive immunotherapy, washed twice, and resuspended in PBS at 5×106 cells/ml.
Detection of T cell proliferative capabilityin vitro
T cells (1×106 cells/ml) were cultured in complete medium in 96-well plates with 5% CO2 at 37°C. TLDCs were added to the T cell culture systems. After a 72-hour incubation, CCK-8 was added to a portion of the wells, with labeling performed as per the manufacturer's protocol (Enzo Life Sciences Inc., Farmingdale, NY, USA). The values of optical density (OD) were detected using a standard microplate reader. The remaining wells received TL-DCs again, and additional CCK-8 was after another 72-hour incubation. The values of OD were detected too.
Detection of interferon-γ (IFN-γ) and interleukin-2 (IL-2) by ELISA
IFN-γ and IL-2 culture concentrations were quantified by ELISA (R&D Systems) according to the manufacturer's protocol.
Fifteen C57BL/6 mice were divided into three groups (those treated with effector cells from naive T cells, effector cells from pooled T cells, or those receiving no treatment as a control group). All mice were injected subcutaneously in the back with 1×105 B16 cells. The mice were pretreated with CTX by peritoneal injection on the day of visible tumor appearance. Cell transfer treatments (1×106 cells/ mouse) were performed on days 13–14 following the tumor cell injections. An investigator blinded to the experiments performed serial tumor measurements on alternate days with calipers, and the tumor volume was calculated (V=1/6τab2).
Detection of CTL homing abilityin vivo
Mice were infused with carboxyfluorescein succinimidyl ester-labeled CTLs. Labeling was performed as per manufacturer's protocol (Invitrogen, Carlsbad, CA, USA), and mice were killed on Day 3 following infusion. Tumors were removed and frozen sections were prepared. The fluorescence intensity of the sections was detected by confocal laser microscopy (Olympus Corporation, Japan).
RNA isolation and quantitative reverse transcriptionpolymerase chain reaction (RT-PCR)
Mice were killed on Day 3 following infusion, and total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions. IL-2, IFN-γ, granzyme B, and perforin mRNA expression were examined. cDNA was synthesized from total RNA (0.5 μg) using PrimeScript RT Kit (Takara Biotechnology, Dalian, China) following the manufacturer's instructions. Subsequently, the cDNA was subjected to real-time PCR using Power SYBR Green PCR Master Mix (Takara Biotechnology). The real-time PCR reaction consisted of 2 μl diluted RT product, 10 μl SYBR Green PCR Master Mix, and 250 nmol/L forward and reverse primers (Table 1) in a total volume of 20 μl. Reactions were carried out on a 7500 real-time PCR System (Applied Biosystems, Foster City, CA, USA) for 40 cycles (95°C for 5 seconds and 60°C for 35 seconds) after an initial 30 seconds incubation at 95°C. The fold change in expression was calculated using the ΔΔCt method with the housekeeping gene β-actin mRNA as an internal control. All experiments were performed in a random, blind fashion at least twice, both of which demonstrated similar results.
All values are presented as mean±standard error (SE). Significance tests were performed using Student's t test or one-way analysis of variance (ANOVA) by SPSS (SPSS, Chicago, IL, USA). Statistical significance was set at P <0.05.
CTX peritoneal injection downregulates Tregs but does not influence tumor regression
Protein levels were significantly reduced after CTX peritoneal injection (Figure 1A and 1B). Flow cytometry was used to quantify the proportion of splenic CD4+CD25+Foxp3+ Tregs. The proportion of Tregs was decreased overall from Days 1 to 14 after injection and reached the lowest level at Day 4 (Figure 1C). Interestingly, there was no significant difference in tumor volume between the groups receiving injections and controls (Figure 1D). Hence, these results suggest that CTX peritoneal pretreatment (100 mg/kg) may change the tumor microenvironment and induce a strong inhibitory immune effect in B16-bearing C57BL/6 mice.
Naive T cells show a stronger proliferative capacity and elevated cytokine production than pooled T cellsin vitro
We tested the proliferative capacity and cytokine secretion capability of TN and TP after being pulsed by DC. There were no significant differences in the proliferative capacity between TN and TP after the primary stimulation, but there was a change following a secondary stimulation (Figure 2A). Following primary stimulation, TP produced greater amounts of IFN-γ and IL-2 than TN. However, TN IFN-γ and IL-2 quantities increased remarkably following secondary stimulation (Figure 2B and 2C). Collectively, these results suggest that TP terminally differentiate and senesce more rapidly than TN.
Effector cells generated from naive T cells mediate more potent antitumor activityin vivo
The efficacy of effector cells derived from TN and TP during tumor treatment was evaluated. For simplicity, effector cells of TN or TP were termed TEFFN and TEFFP, respectively. TEFFN and TEFFP generated from a stimulation of TL-DC were adoptively transferred into pretreated (CTX peritoneal injection) mice bearing established and vascularized tumors (Figure 3A). We examined the homing ability of the effector cells 3 days after infusion. The fluorescence intensity of tumor tissues infused with TEFFN was higher than those infused with TEFFP (Figure 3B). We then measured IL-2, IFN-γ, granzyme, and perforin mRNA levels in tumor tissues using real-time PCR. The mRNA levels in the TEFFN group were remarkably higher than in the TEFFP group (Figure 3C). After a nearly 4-week observation, the change in tumor volume suggests both TEFFN and TEFFP displayed significant antitumor activities. However, TEFFN were significantly more effective than TEFFP (Figure 3D). These data indicate that TEFFN derived effector cells have more proliferative, homing, and cytokines secretion capabilities and mediate superior antitumor immunity in vivo.
It is well established that the immune system plays a crucial role in the defense against tumors. Autologous T lymphocytes produce specific antitumor responses by application of DC presenting tumor antigens. The antitumor effect is mediated primarily by CTLs.1 CTLs carry out specific killing of target cells mediated by Fas molecules (CD95) directly and/or indirectly by secretion of cytokines (IL-2 and IFN-γ). CTLs produce cytokines such as IL-2 and IFN-γ to induce tumor cells to enter apoptosis.4 Research shows that CTLs carry out a marked antitumor effect on tumors with antigens recognized by its T cell receptor (TCR), either in vitro or in normal physiological conditions.5–8
Previous studies have focused mainly on the active immunization mediated by CD8+ CTLs, and how to activate and amplify such cells prior to infusion treatment. However, Dudley et al9 reported that a large number of tumor-specific CTLs were infused into patients, but after a few days could not be detected. This suggested that success criteria of adoptive immunotherapy are not only the number of cells, tumor specificity, affinity, and cellular immune phenotype of the injected tumor, but also in vivo survival time of the input effector cells. How best to maintain an efficient and long-lasting cytotoxic effect has become one of the key topics in tumor immunotherapy. A study by Hinrichs indicated that naive CD8+ CTLs in human and mouse are the predominant cells mediating tumor destruction.10 In recent years, more attention has been paid to CD4+ T cells and their role in anti-infection and antitumor activity.11,12 Studies indicate that CD4+ T cells are required for the generation and maintenance of CD8+ memory T cells and also promote their survival.12–17 The value of naive CD4+ T cells in adoptive immunotherapy has recently been highlighted.18
In this experiment, we separated naive CD8+ T cells and naive CD4+ T cells from splenic suspension using magnetic beads, and then mixed them at a ratio of 2:1 (naive CD4+ T cell: naive CD8+ T cell) to generate the naive T cell. The in vitro experimental data indicated that the proliferative capacity and cytokine secretion of naive T cells were higher than those of the pooled T cells, suggesting that naive T cells could generate more effector cells and secrete more cytokines to induce tumor cell apoptosis. These results further suggest that the killing ability of naive T cellderived CTLs was more powerful than that from pooled T cells. In 1979, Lord formally proposed the concept of the tumor microenvironment.19 The suppressor cells and inhibitory cytokines in the tumor microenvironment constitute the local environment. Sakaguchi first proposed the existence of Tregs in 1995.20 Tregs are a group of cells with immune suppression function and are primarily regulatory T cells. The study showed that in the tumor microenvironment, IL-10 and TGF-β1 induce the body to produce more Treg cells.21,22 Therefore, the levels of IL-10 and TGF-β reflect the state of immune suppression in the local tumor microenvironment. Chemotherapy or radiation therapy changes the tumor microenvironment, and the application of systemic chemotherapy is the most commonly used clinical method that interferes with the tumor microenvironment.23 A low dose of CTX can selectively delete Tregs while retaining an effective number of T cells.24 Our past studies indicated that the most effective CTX dose was 100 mg/kg. In vivo tests suggest that the levels of Treg, TGF-β1, and IL-10 were reduced by peritoneal injection of low-level CTX doses. On the basis of the tumor microenvironment interfering with immune suppression, these results suggest that the homing ability of naive T-derived CTLs is better than that of TP, and the change in tumor volume was significantly different from the TEFFP group. In summary, our results indicate that CTLs derived from TN are more efficient and have a longlasting cytotoxic effect on B16 melanoma mouse models. Although further refinement and validation are necessary, this approach may offer a new strategy for improving cancer immunotherapy.
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